Microparticles useful as ultrasonic contrast agents

ABSTRACT

Microparticles are provided comprising a shell of an outer layer of a biologically compatible material and an inner layer of biodegradable polymer. The core of the microparticles contain a gas, liquid or solid for use in drug delivery or as a contrast agent for ultrasonic contrast imaging. The microparticles are capable of passing through the capillary systems of a subject.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/758,988 filed Jan. 11, 2001, currently pending, which is a divisionalof U.S. application Ser. No. 09/070,474 filed Apr. 30, 1998, now U.S.Pat. No. 6,193,951, which is a continuation-in-part of U.S. applicationSer. No. 08/847,153 filed Apr. 30, 1997, now abandoned, the contents ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Hollow microparticles, sometimes called microbubbles ormicrospheres, are efficient for back scattering ultrasound energy. Thus,small microbubbles injected into the bloodstream, can enhance ultrasonicechographic imaging to aid the visualization of internal structures,such as the heart and blood vessels. The ultrasound contrast is achievedwhen acoustic impedance between two materials at an interface isdifferent. Thus, the greater the difference of acoustic impedancebetween the materials, the greater the intensity of an ultrasound echofrom that interface. Since there is a large difference between theacoustic impedance between body tissue and gas, gas containingmicroparticles circulating within tissue or blood are strong backscatterers of the ultrasound energy. For use in the circulatory system,microparticles should have a diameter of less than about ten microns inorder to pass through the capillaries of the circulatory system. Thelower limit of sufficient echogenicity of a microparticle is about oneto two microns.

[0003] In cardiology, microparticles are useful for intravenousinjection, thereby providing ultrasound contrast in the right chambersof the heart, enhancing identification of cardiac structures, valvefunctions and detection of intracardiac shunts. However in order tovisualize the left chambers of the heart, the microparticles must firstpass through the pulmonary circulation system. Such particles must besmall enough to pass through the pulmonary capillaries. Otherwise theyare trapped within the lungs. They must also have sufficient structuralstrength to survive the pressures within the left chambers of the heart.

[0004] Microparticles also permit the definition of volumes, wallmotion, and other factors that identify diseased states within theheart. The use of contrast agents also facilitates use of Dopplerultrasound techniques because strong echo sources moving in thebloodstream are far more echogenic than red blood cells, which are theusual echo sources used in Doppler ultrasound techniques. Contrastagents in blood may also be used to locate the presence of blood inareas of the body or identify the absence of blood by the lack ofechogenicity in areas that should be echogenic. Examples of such usesare the use of microparticles for assessment of perfusion to themyocardium, and for assessment of defects in the coronary septum by theflow of particles through the septum separating the cardiac chambers.Another example is the use of microparticles to identify vascular embolisuch as blood clots, and abnormal growths into the vascular chambers bythe absence of the ultrasonic contrast.

[0005] Other uses of contrast agents are to examine organ perfusion,such as to assess the damage caused by an infarct, to examine organssuch as the liver, or to differentiate between normal and abnormaltissues, such as tumors and cysts.

[0006] The present invention provides microparticle contrast agentswhich are delivered intravenously but are capable of passing through thepulmonary circulation system for enhanced examination and diagnosis ofboth sides of the heart as well as examination of other tissues andorgans as described above.

[0007] In addition to diagnostic imaging, the microparticles accordingto the present invention are also used for drug delivery where the drugis released from the particle by diffusion from the microparticle, bydegradation of the microparticle, or by rupture of the particle usingultrasonic energy.

SUMMARY OF THE INVENTION

[0008] The present invention provides compositions of microparticles ofwhich the majority of the microparticles have diameters within the rangeof about one to ten microns, have an outer layer comprising abiologically compatible material and an inner layer comprising abiodegradable polymer. The microparticles may have a hollow core,containing either a gas or a liquid, or a solid core.

[0009] The outer layer may be chosen on the basis of biocompatibilitywith the blood stream and tissues, whereas the inner layer may beselected on the basis desired mechanical and acoustic properties. Thematerials of both layers may be selected to predetermine the strength ofthe microparticle, for example to provide a desired resonant frequencyand stability within threshold diagnostic imaging levels of ultrasoundradiation. Methods for forming the multi-layered microparticles and theuse in ultrasonic diagnostic imaging and drug delivery are alsoprovided. The layers may also be chosen by their capability to containand deliver drugs.

BRIEF DESCRIPTION OF THE DRAWINDS

[0010]FIG. 1 is a graph of the time course of the reflected ultrasoundintensity in the left atrium in a test of a contrast agent according toexample 7.

[0011]FIG. 2 is a graph of the time course of the reflected ultrasoundintensity in the left atrium of the contrast agent tested in accordancewith example 8.

[0012]FIG. 3 is a graph of the volumetric size distribution of theunfiltered microcapsules made in example 13, and the size distributionof when the suspension is filtered.

[0013]FIG. 4 shows the resonant frequencies of two microcapsulepreparations having different wall compositions which were tested inaccordance with example 18.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] As used herein the term microparticles is intended to includemicrocapsules, microspheres and microbubbles which are hollow particlesenclosing a core which may be filled with a gas or liquid. It alsoincludes particles in which the core may be a solid material. It is notnecessary for the microparticles to be precisely spherical although theygenerally will be spherical and described as having average diameters.If the microparticles are not spherical, then the diameters are referredto or linked to the diameter of a corresponding spherical microparticlehaving the same mass and enclosing approximately the same volume ofinterior space as a non-spherical microparticle.

[0015] The microparticles according to the present invention have abi-layered shell. The outer layer of the shell will be a biologicallycompatible material or biomaterial since it defines the surface whichwill be exposed to the blood and tissues within the body. The innerlayer of the shell will be a biodegradable polymer, which may be asynthetic polymer, which may be tailored to provide the desiredmechanical and acoustic properties to the shell or provide drug deliveryproperties. For use as ultrasound contrast agents, the cores of themicroparticles contain gas, typically air or nitrogen. However, for drugdelivery purposes the core may either be a liquid or a different solidmaterial from the shell layers. To make the microparticles rupturable bya low intensity ultrasound energy, however, they must contain a gas toallow acoustic coupling and particle oscillation. Microparticles areconstructed herein such that the majority of those prepared in acomposition will have diameters within the range of about one to tenmicrons in order to pass through the capillary system of the body.

[0016] Since the microparticles have an outer and inner layer, thelayers can be tailored to serve different functions. The outer shellwhich is exposed to the blood and tissues serves as the biologicalinterface between the microparticles and the body. Thus it will be madeof a biocompatible material which is typically amphiphilic, that is, hasboth hydrophobic and hydrophilic characteristics. Blood compatiblematerials are particularly preferred. Such preferred materials arebiological materials including proteins such as collagen, gelatin orserum albumins or globulins, either derived from humans or having astructure similar to the human protein, glycosoaminoglycans such ashyaluronic acid, heparin and chondroiten sulphate and combinations orderivatives thereof. Synthetic biodegradable polymers, such aspolyethylene glycol, polyethylene oxide, polypropylene glycol andcombinations or derivatives may also be used. The outer layer istypically amphiphilic, as well as having a chemistry which allows chargeand chemical modification. The versatility of the surface allows forsuch modifications as altering the charge of the outer shell, such as byselecting a type A gelatin having an isoelectric point abovephysiological pH, or by using a type B gelatin having an isoelectricpoint below physiological pH. The outer surfaces may also be chemicallymodified to enhance biocompatibility, such as by PEGylation,succinylation or amidation, as well as being chemically binding to thesurface targeting moiety for binding to selected tissues. The targetingmoieties may be antibodies, cell receptors, lectins, selecting,integrins or chemical structures or analogues of the receptor targets ofsuch materials. The mechanical properties of the outer layer may also bemodified, such as by cross linking, to make the microparticles suitablefor passage to the left ventricle, to provide a particular resonantfrequency for a selected harmonic of the diagnostic imaging system, orto provide stability to a threshold diagnostic imaging level of theultrasound radiation.

[0017] The inner shell will be a biodegradable polymer, which may be asynthetic polymer. An advantage of the inner shell is that it providesadditional mechanical or drug delivery properties to the microparticlewhich are not provided or insufficiently provided by the outer layer, orenhances mechanical properties not sufficiently provided by the outerlayer, without being constrained by surface property requirements. Forexample, a biocompatible outer layer of a cross-linked proteinaceoushydrogel can be physically supported using a high modulus syntheticpolymer as the inner layer. The polymer may be selected for its modulusof elasticity and elongation, which define the desired mechanicalproperties. Typical biodegradable polymers include polycaprolactone,polylactic acid, polylactic-polyglycolic acid co-polymers, co-polymersof lactides and lactones, such as epsilon-caprolactone,delta-valerolactone, polyalkylcyanoacrylates, polyamides,polyhydroxybutryrates, polydioxanones, poly-beta-aminoketones,polyanhydrides, poly-(ortho) esters, polyamino acids, such aspolyglutamic and polyaspartic acids or esters of polyglutamic andpolyaspartic acids. References on many biodegradable polymers are citedin Langer, et. al. (1983) Macromol.Chem.Phys.C23, 61-125.

[0018] The inner layer permits the modification of the mechanicalproperties of the shell of the microparticle which are not provided bythe outer layer alone. Moreover, the inner layer may provide a drugcarrier and/or drug delivery capacity which is not sufficient orprovidable by the outer layer alone. For use as an ultrasonic contrastagent, the inner layer will typically have thickness which is no largerthan is necessary to meet the minimum mechanical or drugcarrying/delivering properties, in order to maximize the interior gasvolume of the microparticle. The greater the gas volume within themicroparticle the better the echogenic properties.

[0019] The combined thickness of the outer and inner layers of themicroparticle shell will depend in part on the mechanical and drugcarrying/delivering properties required of the microparticle, buttypically the total shell thickness will be in the range of 25 to 750nm.

[0020] The microparticles may be prepared by an emulsification processto control the sequential interfacial deposition of shell materials. Dueto the amphiphilicity of the material forming the outer layer, stableoil/water emulsions may be prepared having an inner phase to outer phaseratio approaching 3:1, without phase inversion, which can be dispersablein water to form stable organic phase droplets without the need forsurfactants, viscosity enhancers or high shear rates.

[0021] Two solutions are prepared, the first being an aqueous solutionof the outer biomaterial. The second is a solution of the polymer whichis used to form the inner layer, in a relatively volatilewater-immiscible liquid which is a solvent for the polymer, and arelatively non-volatile water-immiscible liquid which is a non-solventfor the polymer. The relatively volatile water-immiscible solvent istypically a C5-C7 ester, such as isopropyl acetate. The relativelynon-volatile water-immiscible non-solvent is typically a C6-C20hydrocarbon such as decane, undecane, cyclohexane, cyclooctane and thelike. In the second solution containing the polymer for the inner layer,the polymer in water-immiscible solvents are combined so that thepolymer fully dissolves and the two solvents are miscible withagitation. The polymer solution (organic phase) is slowly added to thebiomaterial solution (aqueous phase) to form a liquid foam. Typicallyabout three parts of the organic polymer solution having a concentrationof about 0.5 to 10 percent of the polymer is added to one part of theaqueous biomaterial solution having a concentration of about 1 to 20percent of the biomaterial. The relative concentrations of the solutionsand the ratio of organic phase to aqueous phase utilized in this stepessentially determine the size of the final microparticle and wallthickness. After thorough mixing of the liquid foam, it is dispersedinto water and typically warmed to about 30-35° C. with mild agitation.While not intending to be bound by a particular theory, it is believedthat the biomaterial in the foam disperses into the warm water tostabilize an emulsion of the polymer in the organic phase encapsulatedwithin a biomaterial envelope. To render the biomaterial envelope waterinsoluble, a cross linking agent, such as glutaraldehyde, is added tothe mixture to react with the biomaterial envelope and render it waterinsoluble, stabilizing the outer shell. Other cross-linking agents maybe used, including the use of carbodiimide cross-linkers.

[0022] Since at this point the inner core contains a solution of apolymer, a solvent and a non-solvent with different volatilities, as themore volatile solvent evaporates, or is diluted, the polymerprecipitates in the presence of the less volatile non-solvent. Thisprocess forms a film of precipitate at the interface with the innersurface of the biomaterial shell, thus forming the inner shell of themicroparticle after the more volatile solvent has been reduced inconcentration either by dilution, evaporation or the like. The core ofthe microparticle then contains predominately the organic non-solvent.The microparticles may then be isolated by centrifugation, washed,formulated in a buffer system, if desired, and dried. Typically, dryingby lyophilization removes not only the non-solvent liquid core but alsothe residual water to yield gas-filled hollow microparticles.

[0023] It may be desirable to further modify the surface of themicroparticle, for example, in order to passivate surfaces againstmacrophages or the reticuloendothelial system (RES) in the liver. Thismay be accomplished, for example by chemically modifying the surface ofthe microparticle to be negatively charged since negatively chargedparticles appear to better evade recognition by macrophages and the RESthan positively charged particles. Also, the hydrophilicity of thesurface may be changed by attaching hydrophilic conjugates, such aspolyethylene glycol (PEGylation) or succinic acid (succinylation) to thesurface, either alone or in conjunction with the charge modification.

[0024] The biomaterial surface may also be modified to provide targetingcharacteristics for the microparticle. The surface may be tagged byknown methods with antibodies or biological receptors. For example, ifthe microparticle were treated to target tumors and were hollow, theycould be used for ultrasound detection to enhance diagnosis of thetumors. If the microparticles were filled with drugs they could be usedto target the tumors where the drug could be preferentially released atthe target site, for example, by increasing the ultrasonic energy torupture the particles at the appropriate time and location.

[0025] The microparticles may also be sized or processed aftermanufacture. This is an advantage over lipid-like microparticles whichmay not be subjected to mechanical processing after they are formed dueto their fragility.

[0026] The final formulation of the microparticles after preparation,but prior to use, is in the form of a lyophilized cake. The laterreconstitution of the microparticles may be facilitated bylyophilization with bulking agents which provide a cake having a highporosity and surface area. The bulking agents may also increase thedrying rate during lyophilization by providing channels for the waterand solvent vapor to be removed. This also provides a higher surfacearea which would assist in the later reconstitution. Typical bulkingagents are sugars such as dextrose, mannitol, sorbitol and sucrose, andpolymers such as PEG's and PVP's.

[0027] It is undesirable for the microparticles to aggregate, eitherduring formulation or during later reconstitution of the lyophilizedmaterial. Aggregation may be minimized by maintaining a pH of at leastone to two pH units above or below the isoelectric point(P_(i)) of thebiomaterial forming the outer surface. The charge on the surface isdetermined by the pH of the formulation medium. Thus, for example, ifthe surface of the biomaterial has a P_(i) of 7 and the pH of theformulation medium is below 7, the microparticle will possess a netpositive surface charge. Alternatively, if the pH of the formulationmedium is greater than 7, the microparticle would possess a negativecharge. The maximum potential for aggregation exist when the pH of theformulation medium approaches the P_(i) of the biomaterial used in theouter shell. Therefore by maintaining a pH of the formulation medium atleast one to two units above or below the P_(i) of the surface,microparticle aggregation will be minimized. As an alternative, themicroparticles may be formulated at or near the P_(i) with the use ofsurfactants to stabilize against aggregation. In any event, buffersystems of the final formulation to be injected into the subject shouldbe physiologically compatible.

[0028] The bulking agents utilized during lyophilization of themicroparticles may also be used to control the osmolality of the finalformulation for injection. An osmolality other than physiologicalosmolality may be desirable during the lyophilization to minimizeaggregation. However, when formulating the microparticles for use, thevolume of liquid used to reconstitute the microparticles must take thisinto account.

[0029] Other additives may be included in order to prevent aggregationor to facilitate dispersion of the microparticles upon formulation.Surfactants may be used in the formulation such as poloxomers(polyethylene glycol-polypropylene glycol-polyethylene glycol blockco-polymers). Water soluble polymers also may assist in the dispersionof the microparticles, such as medium molecular weightpolyethyleneglycols and low to medium molecular weightpolyvinylpyrolidones.

[0030] If the formulation is to contain a drug-containing core, themicroparticles may be soaked in a solution of the drug whereby thesolution diffuses into the interior. In particular, the use of bilayeredmicroparticles where the inner shell has a porous characteristic allowsfor rapid diffusion of a drug solution into the hollow core. Themicroparticles may be re-dried such as by lyophilization to produce agas filled, drug containing microparticle. The combination of the drugwith prefabricated particles allows one to avoid processing which maylead to drug degradation. To provide microparticles having a solid corecontaining a drug, during formation of the microparticles, the thicknessof the inner layers may be increased to occupy more or all of theinterior volume. Then, by later soaking in the drug-containing solution,the inner solid core will absorb the drug and provide a solid reservoirfor the drug. Alternatively, the drug may be dissolved in the organicphase with the biopolymer during the microparticle forming process.Evaporation of the organic solvents causes the drug to coprecipitatewith the biopolymer inside the microparticle.

[0031] It will be realized that various modifications of theabove-described processes may be provided without departing from thespirit and scope of the invention. For example, the wail thickness ofboth the outer and inner layers may be adjusted by varying theconcentration of the components in the microparticle-forming solutions.The mechanical properties of the microparticles may be controlled, notonly by the total wall thickness and thicknesses of the respectivelayers, but also by selection of materials used in each of the layers bytheir modulus of elasticity and elongation, and degree of cross-linkingof the layers. Upon certain conditions involving rapid deposition of theinner polymer or very low inner polymer content porosity of the innerpolymer shell is observed. The pores range from approximately 0.1 to 2micron in diameter as observed under electron microscopy. Mechanicalproperties of the layers may also be modified with plasticizers or otheradditives. Adjustment of the strength of the shell may be modified, forexample, by the internal pressure within the microparticles. Preciseacoustical characteristics of the microparticle may be achieved bycontrol of the shell mechanical properties, thickness, as well as narrowsize distribution. The microparticles may be ruptured by ultrasonicenergy to release gases trapped within the microparticles into the bloodstream. In particular, by appropriately adjusting the mechanicalproperties, the particles may be made to remain stable to thresholddiagnostic imaging power, while being rupturable by an increase in powerand/or by being exposed to its resonant frequency. The resonantfrequency can be made to be within the range of transmitted frequenciesof diagnostic body imaging systems or can be a harmonic of suchfrequencies. During the formulation process the microparticles may beprepared to contain various gases, including blood soluble or bloodinsoluble gases. It is a feature of the invention that microparticlecompositions may be made having a resonant frequency greater or equal to2 MHz, and typically greater or equal to 5 MHz.

[0032] Typical diagnostic or therapeutic targets for microparticles ofthe invention are the heart and tumors.

[0033] The following examples are provided by way of illustration butare not intended to limit the invention in any way.

EXAMPLE 1 Preparation of Gelatin Polycaprolactone Microparticles

[0034] A solution of 1.0 gms gelatin (275 bl, isoelectric point of 4.89)dissolved in 20 ml deionized water was prepared at approximately 60 C.Native pH of the solution was 5.07. Separately, 1.0 gms polycaprolactone(M.W. 50,000) and 6.75 ml cyclooctane was dissolved in 42 ml isopropylacetate with stirring at approximately 70 C. After cooling to 37 C, theorganic mixture was then slowly incorporated into the gelatin solutionmaintained at 30 C and under moderate shear mixing using a rotary mixer.Once the organic phase was fully incorporated, the mixing rate wasincreased to 2,500 rpm for 5 minutes and then stirred at low shear foran additional 5 minutes. The resulting o-w emulsion was then added withstirring to 350 ml deionized water maintained at 30 C and containing 1.2ml 25% gluteraldehyde. Immediately after the addition of the emulsion,the bath pH was adjusted to 4.7. After 30 minutes, the pH was adjustedto 8.3. Low shear mixing was continued for approximately 2½ hours untilthe isopropyl acetate had completely volatilized. Polyoxamer 188 in theamount of 0.75 gm was then dissolved into the bath. The resultingmicroparticles were retrieved by centrifugation and washed 2 times in anaqueous solution of 0.25% polyoxamer 188.

[0035] Microscopic inspection of the microparticles revealed sphericalcapsules having a thin-walled polymer shell encapsulating a liquidorganic core. Staining the slide preparation with coomassie blue Gindicated the presence of an outer protein layer uniformly surroundingthe polymer shell.

[0036] The particle size spectrum was determined using a Malvern Micro.Median diameter was 4.78 microns with a spectrum span of 0.94.

EXAMPLE 2 Preparation of Contrast Agent Formulation

[0037] A quantity of microparticles prepared in a manner similar toexample 1 were suspended into an aqueous solution of 25 mM glycine, 0.5%pluronic f-127, 1.0% sucrose, 3.0% mannitol, and 5.0% PEG-3400. Thesuspension was then lyophilized. The resulting dry powder wasreconstituted in deionized water and examined under the microscope toreveal that the microparticles now contained a gaseous core. Stainingthe preparation with commassie blue G confirmed that the outer proteinlayer surrounding the capsules was intact and had survived thelyophilization process.

[0038] Echogenicity was confirmed by insonating at both 2½ and 5 MHz aquantity of lyophilized microparticles dispersed in 120 ml deionizedwater. Measurement was taken at least 15 minutes after dispersion of themicrocapsules to insure that the back scattered signal was due solelyfrom the gas contained within the microparticles. The B mode displayshowed a high contrast indicating that the microparticles were gasfilled.

EXAMPLE 3 Preparation of Gelatin Polylactide Microparticles

[0039] A solution of 1.2 gm gelatin (225 bloom, isoelectric point of5.1) dissolved in 20 ml deionized water was prepared at approximately 50C. Solution pH was adjusted to 6.1 using 1 M NaOH. Separately, 0.07 gmsparaffin, 4.5 ml decane, and 0.69 gms poly DL-lactide (inherentviscosity of 0.69 dL/gm in CHCl₂ @ 30 C) was dissolved into 37 mlisopropyl acetate. The organic mixture was then slowly incorporated intothe gelatin solution which was being maintained at 30 C under moderateshear mixing using a rotary mixer. Once the organic phase was fullyincorporated, the mixing rate was increased to 2,000 rpm for 2 minutesand then reduced to approximately 1,000 rpm for 4 minutes. The resultingliquid foam was mixed into 350 ml deionized water maintained at 30 C and1 ml 25% gluteraldehyde was then added dropwise. Rotary mixing wascontinued for approximately 3 hours until the isopropyl acetate hadvolatilized. The resulting microparticles were retrieved bycentrifugation and washed 2 times in an aqueous solution of 0.25%pluronic f-127.

[0040] Microscopic inspection revealed hollow spherical microparticleshaving an outer protein layer and an inner organic liquid core.

[0041] The microparticles were lyophilized and tested in a mannersimilar to example 2. The results confirmed that the microparticlescontained a gaseous core and were strongly echogenic.

EXAMPLE 4 Preparation of Gelatin Polycaprolactone Microparticles

[0042] A solution of 1.0 gm gelatin (225 bloom, isoelectric point of5.1) dissolved in 20 ml deionized water was prepared at approximately 60C. Solution pH was 4.8. Separately, 0.57 gms polycaprolactone (M.W.50,000) was dissolved into 1.72 ml tetrahydrofuran. To this was addedwith stirring a mixture of 0.07 gms paraffin, 0.475 gm triethyl citrate,4.5 ml cyclooctane, and 42 ml isopropyl acetate. The organic mixture wasthen slowly incorporated into the gelatin solution which was maintainedat 30 C and under moderate shear mixing using a rotary mixer. Once theorganic phase was fully incorporated, the mixing rate was increased to4,700 rpm for 2 minutes and then reduced to 2,000 rpm for 4 minutes. Theresulting liquid foam was then added with stirring to 350 ml of 30 Cdeionized water. To crosslink the gelatin, 1 ml of 25% glutaraldehydewas added dropwise. Mixing was continued for approximately 3 hours untilthe isopropyl acetate had volatilized. The resulting microparticles wereretrieved by centrifugation and washed 2 times in a 0.25% pluronic f-127solution.

[0043] Microscopic inspection revealed discrete hollow spherical polymermicroparticles having an outer protein layer and an inner organic liquidcore.

[0044] The microparticles were lyophilized and tested in a mannersimilar to example 2. The results confirmed that the microparticlescontained a gaseous core and were strongly echogenic.

EXAMPLE 5 Preparation of Gelatin Polycaprolactone Microparticles withCardodiimide Cross-linking

[0045] A solution of 1.0 grams gelatin (225 bloom, isoelectric point of5.1) dissolved into 20 ml deionized water was prepared at approximately60 C. Solution pH was adjusted to 5.5 with 1 M NaOH. Separately, 0.85gms polycaprolactone (M.W. 80,000) was dissolved in 2.5 mltetrahydrofuran. To this was added with stirring a mixture of 0.07 gmsparaffin, 4.5 ml cyclooctane and 42 ml isopropyl acetate. The organicmixture was then slowly incorporated into the gelatin solution which wasmaintained at 30 C and under moderate shear mixing using a rotary mixer.Once the organic phase was fully incorporated, the mixing rate wasincreased to 3,500 rpm for 6.minutes and then reduced to 3,000 rpm for 4minutes. The resulting liquid foam was then dispersed with low shearmixing into 350 ml of a 0.5 M NaCl solution maintained at 30 C. Gelatincrosslinking was accomplished by the slow addition of 200 mg of1-ethyl-3-(3-dimethylamino-propyl) carbodiimide dissolved in 3.0 mldeionized water. Mixing was continued for approximately 3 hours untilthe isopropyl acetate had volatilized. The resulting microparticles wereretrieved by centrifugation and washed 2 times in an aqueous solution of0.25% Pluronic f-127.

[0046] Microscopic inspection revealed discrete hollow spherical polymermicroparticles having an outer protein layer and an inner organic liquidcore.

EXAMPLE 6 Preparation of Surface PEGylated Microparticles

[0047] Microcapsules were prepared in a manner similar to example 1.After centrifugation the cream (approximately 15 ml) was retrieved anddispersed into a solution of 65 ml deionized water, 0.50 gmsmethoxy-PEG-NCO (M.W. 5000), and 0.50 ml triethylamine. After allowingthe mixture to react overnight at room temperature and with mildagitation, the capsules were retrieved by centrifugation and washed 3times in a neutrally buffered solution of 0.25% Pluronic f-127.

EXAMPLE 7 Canine Study of Echogenicity

[0048] One vial of lyophilized microparticles prepared in Example 2 werereconstituted using water. A transesophageal ultrasound probe waspositioned in the esophagus of an anesthetized dog such that afour-chamber view of the heart was obtained. The microparticlesuspension was injected into the femoral vein of the dog. The appearanceof the contrast agent was clearly noted in the ultrasound image of theright chambers of the heart. Subsequently, the agent was observed in theleft chambers of the heart indicating the passage through the capillarysystem of the lungs. The time-course of the reflected ultrasoundintensity in the left atrium was determined by video densitometry. Theagent was seen to persist in the left chambers of the heart forapproximately 6 minutes (FIG. 1).

EXAMPLE 8 Canine Study of Echogenicity Using PEGylated Microparticles

[0049] One vial of lyophilized microparticles prepared in Example 6 wasreconstituted using water. A transesophageal ultrasound probe waspositioned in the esophagus of an anesthetized dog such that afour-chamber view of the heart was obtained. The microparticlesuspension was injected into the femoral vein of the dog. The appearanceof the contrast agent was clearly noted in the ultrasound image of theright chambers of the heart. Subsequently, the agent was observed in theleft chambers of the heart indicating the passage through the capillarysystem of the lungs. The time-course of the reflected ultrasoundintensity in the left atrium was determined by video densitometry. Theagent was seen to persist in the left chambers of the heart forapproximately 16 minutes (FIG. 2) after which time no further data wascollected.

EXAMPLE 9 Preparation of Albumin Polycaprolactone Microparticles

[0050] A 6% aqueous solution was prepared from a 25% solution of USPgrade human serum albumin (Alpha Therapeutic Corp) by dilution withdeionized water. The solution was adjusted to a pH of 3.49 using 1 NHCl. Separately, 8 parts by weight polycaprolactone (M.W. 50,000) and 45parts cyclooctane were dissolved in 300 parts isopropyl acetate atapproximately 70 C. Once dissolution was complete, the organic solutionwas allowed to cool to 37 C. With mild stirring, 42.5 gm of the preparedorganic solution was slowly incorporated into 25.0 gm of the albuminsolution while the mixture was maintained at 30 C. The resulting coarseo-w emulsion was then circulated through a stainless steel sinteredmetal filter element having a nominal pore size of 7 microns.Recirculation of the emulsion was continued for 8 minutes. The emulsionwas then added with stirring to 350 ml deionized water maintained at 30C and containing 1.0 ml of 25% gluteraldehyde. During the addition, thepH of the bath was monitored to insure that it remained between 7 and 8.Final pH was 7.1. Low shear mixing was continued for approximately 2½hours until the isopropyl acetate had completely volatilized. Poloxamer188 in the amount of 0.75 gm was then dissolved into the bath. Theresulting microparticles were retrieved by centrifugation and washed 2times in an aqueous solution of 0.25% poloxamer.

[0051] Microscopic inspection of the suspension revealed sphericalparticles having a thin-walled polymer shell with an outer protein layerand an organic liquid core. The peak diameter as, determined by theMalvern Micro particle size analyzer, was 4.12 microns.

[0052] The suspension was then lyophilized in a manner similar to thatdescribed in Example 2. The resulting dry cake was reconstituted withdeionized water and examined under the microscope to reveal that themicroparticles were spherical, discrete, and contained a gaseous core.

EXAMPLE 10 Protein Content of Microparticles

[0053] Microparticles were prepared in accordance with example 9. Aftercentrifugation approximately 1 ml of the microparticle containing creamwas retrieved and diluted 10 to 1 using deionized water. From thediluted cream, 20 microliter samples were then prepared in triplicate at1×, 2×, and 4× dilutions with deionized water. Protein content of thesamples were determined using a Pierce calorimetric BCA assay and abovine serum albumin standard. Average total protein of the dilutedcream was determined to be 0.441 mg/ml. To determine the total dryweight of the diluted cream, 2 ml were dried in a 40 C oven until nofurther weight change was observed (approximately 16 hours). The averageweight of 4 replicates was 6.45 mg/ml. The percent dry weight of proteinwhich can be used as a measure of the ratio of the protein outer layerto the polymer inner layer of the microcapsule wall can be determinedwith the following formula.

Average total protein/ml÷dry weight/ml×100%

[0054] The percent dry weight of protein was calculated to be 6.8%.

EXAMPLE 11 Preparation of Albumin Polylactide Microparticles

[0055] A 6% aqueous solution was prepared from a 25% solution of USPgrade human albumin by dilution with deionized water. Ion exchange resin(AG 501-X8, BioRad Laboratories) was then added to the solution at aratio of 1.5 gm resin to 1.0 gm dry weight of albumin. After 3 hours theresin was removed by filtration and the pH of the solution was adjustedfrom 4.65 to 5.5 Separately, 0.41 gm d-l lactide (0.69 dL/gm in CHCl₃:at 30 C) and 5.63 gm cyclooctane were dissolved in 37.5 gm isopropylacetate. The organic solution was then slowly incorporated into 25.0 gmof the prepared albumin solution with mild stirring while the mixturewas maintained at 30 C. The resulting coarse o-w emulsion was thencirculated through a stainless steel sintered metal filter elementhaving a nominal pore size of 7 microns. Recirculation of the emulsionwas continued for 8 minutes. The emulsion was then added with stirringto 350 ml deionized water maintained at 30 C and containing 1.0 ml of25% gluteraldehyde. During the addition, the pH of the bath wasmonitored to insure that it remained between 7 and 8. Final pH was 7.0.Low shear mixing was continued for approximately 2½ hours until theisopropyl acetate had completely volatilized. Polyoxamer 188 in theamount of 0.75 gm was then dissolved into the bath. The resultingmicrospheres were retrieved by centrifugation and washed 2 times in anaqueous solution of 0.25% polyoxamer.

[0056] Microscopic inspection revealed hollow spherical polymermicroparticles having an outer protein layer and an inner organic liquidcore. The suspension was formulated with a glycine/PEG 3350 excipientsolution, then lyophilized. The resulting dry cake was reconstitutedwith deionized water and examined under the microscope to reveal thatthe microparticles were spherical, discrete, and contained a gaseouscore.

EXAMPLE 12 PEG Modification of the Microparticle Surface

[0057] Microparticles were prepared in a manner similar to example 9.After centrifugation, 4 ml of the microparticles containing cream(approximately 11 ml total yield) was resuspended in 31 ml deionizedwater. To this was added a 10 ml solution containing 0.3 gmmethoxy-peg-NCO 5000 and the pH was adjusted to 8.7. The mixture wasallowed to react at room temperature with mild agitation for 4½ hours.At the end of this period the pH was measured to be 7.9. Themicroparticles were retrieved by centrifugation and washed 2 times in a0.25% solution of polyoxamer 188. The suspension was formulated with aglycine/PEG 3350 excipient solution, then lyophilized. The resulting drycake was reconstituted with deionized water and examined under themicroscope to reveal that the microparticles were spherical, discrete,and contained a gaseous core.

EXAMPLE 13 Post-Fabrication, Modification of Size Distribution

[0058] A quantity of microparticles were first prepared in a mannersimilar to example 1 with procedures modified to provide a broadenedsize spectrum. After washing and retrieval by centrifugation roughlyhalf the microparticle containing cream was diluted to 125 ml with a0.25% solution of polyoxamer 188. The suspension was then filtered usinga 5 micron sieve type pc membrane filter (Nuclepore) housed in a stirredcell (Amicon). The retentate was discarded while the permeate was againfiltered using a 3 micron sieve type filter in the stirred cell systemuntil the retentate volume reached approximately 20 ml. The retentatewas diluted to a volume of 220 ml using 0.25% polyoxamer 188 solution.The 3 micron filtration process was repeated until the retentate volumewas again approximately 20 ml.

[0059]FIG. 3 provides a comparison of the volumetric size distributionof the unfiltered microparticle suspension with the 5 micron permeateand the 3 micron retentate. The results, derived from a Malvern Microparticle size analyzer show a stepwise narrowing of the size spectrumtoward a specific size range defined by the pore size of the filtersused.

EXAMPLE 14 Representative Canine Study of Echogenicity

[0060] A 31 kg, thoracotomized male mongrel dog was injected with 1 ccof reconstituted microparticle composition made according to example 4.This was delivered to the circulation through a peripheral venousinjection. Triggered harmonic ultrasound imaging (once every beat) ofthe left ventricle was performed for 9 minutes. A contrast effect couldbe seen in the myocardium during triggered imaging. Real-time (30 Hz)harmonic ultrasound imaging over the next 4 minutes increased bubbledestruction. Left ventricular opacification remained persistent over the13-minute imaging period. No adverse hemodynamic effects were observed.

[0061] In a separate study, 0.1 cc of reconstituted microparticle agentwas administered similarly to a thoracotomized male mongrel dog.Triggered harmonic ultrasound imaging was performed for 1 minute,followed by 4 minutes of increased microparticle destruction withreal-time imaging. Again, no adverse hemodynamics effects were seen, andleft ventricular opacification was apparent and persistent.

EXAMPLE 15 Dye Loading of Albumin Polylactide Microparticles

[0062] A lyophilized cake in a 10 ml serum vial, composed of excipientand lactide-containing microparticles prepared in a manner similar toExample 11 was placed into a 50 ml centrifuge tube. Enough isopropylalcohol was added to cover the cake and it was allowed to soak for 30seconds. Aqueous Pluronic F68 solution (0.25% w/w) was added to fill thetube. After centrifuging, the supernatant was removed and another rinseperformed. A saturated, filtered solution of rhodamine B was added tothe microparticles and allowed to soak overnight. Under the microscope,the microparticles appeared filled with dye solution. A dye saturatedF68 solution was made to use as a lyophilization excipient. Four ml ofthe excipient was combined with the approximately 2 ml of microcapsulecontaining solution and the resulting mixture was split between two 10ml serum vials. The vials were frozen at −80° C. and lyophilized in aFTS tray dryer. Both vials were purged with perfluorobutane gas by fivepump-down purge cycles with a vacuum pump. Observation showed somemicroparticles that were half full of red solution and half full of gas.There was no obvious leakage of the dye from these microparticles duringobservation. The microparticles were rinsed with four, 20 ml portions ofF68 solution on a vacuum filter. The microparticles were placed in acuvette, centrifuged, and an initial spectra was taken. The cuvette wassonicated in an ultrasonic bath, centrifuged, and another spectra taken.Abs. Initial (553-800) Abs. Sonicated (553-800) 1.164 1.86

[0063] The higher absorption after sonication indicates thatencapsulated dye was released upon insonation of the microparticles.

EXAMPLE 16 Preparation of Wall Modified Albumin PolycaprolactoneMicroparticles

[0064] Albumin coated microcapsules were prepared in a manner similar toexample 9 with the exception that 0.20 gm paraffin was also dissolvedinto the organic solution along with the polycaprolactone and thecyclooctane.

[0065] Microscopic inspection of the finished microparticle suspensionrevealed spherical particles having a morphology and appearancevirtually identical to those prepared without the addition of paraffin.

EXAMPLE 17 Dye Loading of Human Serum Albumin PolycaprolactoneMicroparticles

[0066] A lyophilized cake in, a 10 ml serum vial, composed of excipientand paraffin-containing microparticles prepared in accordance withexample 16 was placed into a 50 ml centrifuge tube. The cake was coveredwith methanol and allowed to soak for 30 seconds. The tube was thenfilled with an aqueous solution of 0.25% (w/w) Pluronic F68, gentlymixed, and centrifuged in order to precipitate the now fluid-filledmicrocapsules. The supernatant was removed and the tubes were againfilled with pluronic solution. The microparticles were resuspended byvortexing and again centrifuged. After removing the supernatantsolution, two ml of a saturated, filtered solution of brilliant blue Gdye in 0.25% (w/w) aqueous F68 was added. The microparticles wereallowed to soak for approximately 72 hours. Microscopic examinationrevealed 90-95% of the microparticles to be filled with dye solution. Alyophilization excipient was prepared. Four ml of the excipient wasadded to the microparticle solution and mixed by vortexing. Two 10 mlserum vials were filled with 3 ml each of solution and frozen at −80° C.The vials were lyophilized on a FTS flask lyophilizer. Both vials and aportion of deionized water were purged with perfluorobutane for 10minutes. Both vials were reconstituted with deionized water and rinsedwith two 40 ml portions of 0.25% (w/w) F68 solution on a vacuum filter.The resulting microparticle solution was split into two 3 ml portions.One portion was sonicated in an ultrasonic bath to rupture the bubbles.Both portions were diluted 1/10 with F68 solution and placed intoUV-visible cuvettes. The cuvettes were centrifuged and a visible spectrawas taken.

[0067] Absorption (at 605 nm-800 nm)

[0068] Sonicated 0.193

[0069] Non-sonicated 0.136

[0070] The higher absorption after sonication indicates thatencapsulated dye was released upon insonation of the microcapsules.

EXAMPLE 18 Acoustic Resonance of Microparticles

[0071] To demonstrate a method of acoustically tuning the microparticleconstruct, microparticles prepared in accordance with the proceduresdescribed in examples 9 and 16 were reconstituted with deionized waterand compared for their acoustic properties using procedures described asfollows:

[0072] Two matched 5 MHz transducers were placed in a tank filled withdegassed water facing one another. Water depth was approximately 3inches. The transducers, one an emitter and the other a receiver, werepositioned 6 inches apart to maximize the received signals. A 2 inchdiameter, 2 cm wide circular chamber was placed between the twotransducers with the mid chamber position at 3 inches from the emitter.The two circular faces of the chamber were covered with 3 milpolyethylene film and the chamber was then filled with degassed water.Sound waves readily propagated from the emitter through the chamber tothe receiver. The sound source was set to Gaussian Noise with 10 Voltpeak to peak amplitude output from the ultrasound generator. Thereceiver signal is amplified with a 17 dB preamp and an oscilloscope.The oscilloscope digital electronics can perform Fast Fourier Transforms(FFT) of the received wave forms and display these distributions. Afterbaseline readings were made, test microparticle contrast materials weredelivered within the chamber via hypodermic syringe and thoroughly mixedtherein by pumping the syringe. During post-test evaluation, the FFTdata was converted into the Transfer Function of the test agent.

[0073] The Transfer Function (TF) is determined by dividing the bubbletransmission spectral data by the spectral data without bubbles, i.e:

TF=T(f)_(with bubbles) /T(f)_(no bubbles)

[0074] where T(f) is requested by the FFT.

[0075] The contrast agent selectively attenuates sound waves dependingupon its spectral distribution, i.e. more sound energy is absorbed at ornear bubble resonance than off-resonance. Thus the procedure can be usedto assess the resonant spectral distribution of the agent.

[0076] Data derived from the two agents with nearly identical sizedistribution but different inner shell thickness were collected on thesame day with the same equipment set at the same settings. Everythingelse was held constant for a variety of agent dosages.

[0077] Normalization of the spectra was performed by dividing thespectral array by the minimal value. Thus the peak value becomes unityand when plotted on the same graph it becomes quite easy todifferentiate the two graphs. These normalized data are presented inFIG. 4.

[0078] Inspection of the results shown in FIG. 4 clearly show that whenshell wall compliance is increased, the resonant frequency can be madeto shift from 2.3 MHz to 8.9 MHz. Thus, the resonant frequency of anagent can be controlled by controlling the wall composition andthickness.

EXAMPLE 19 Effect of Acoustic Properties on in-vivo Echogenicity

[0079] Two air-containing microparticle formulations were evaluated forefficacy in-vivo. One vial of lyophilized microparticles was prepared asdescribed in Examples 1 and 2 (formulation A). A second vial oflyophilized microparticles were prepared in a manner similar to thatdescribed in Examples 1 and 2 except that four times the amount ofpolymer was used, yielding microcapsules with a thick inner wall andhence a higher resonant frequency (formulation B). Both vials werereconstituted immediately prior to use. From particle size analysis,both formulations had a mean microparticle diameter of approximately 4microns and nearly identical microparticle concentration. In-vitroacoustical characterization showed formulation A to have a resonantfrequency near 5 MHz, and formulation B to have a resonant frequencygreater than 10 MHz. A 5 MHz transesophageal ultrasound probe waspositioned in the esophagus of an anesthetized dog such that afour-chamber view of the heart was obtained. The reconstitutedmicroparticle suspension (4 cc of formulation A) was injected into thefemoral vein of the dog. The appearance of the contrast agent wasclearly noted in the ultrasound image of the right and left chambers ofthe heart. Subsequently, 4 cc of the thick walled microparticlesuspension (formulation B) was injected into the femoral vein of thedog. While the appearance of the contrast agent was again clearly notedin the ultrasound image of the heart, the contrast effect wassubstantially diminished when compared to the equivalent volumeinjection of formulation A. Subsequent injections of dilutions made fromformulation A demonstrated a greater than four fold dose effectivenessof formulation A which had a resonant frequency near the centerfrequency of the ultrasound diagnostic system as compared to formulationB with a greater peak resonant frequency.

What is claimed is:
 1. A composition comprising: a plurality ofmicroparticles, a majority of which have diameters in the range of about1 to 10 micrometers, each of which comprises a shell enclosing agas-filled hollow core, said shell comprising an outer layer of across-linked amphiphilic material and an inner layer of a biodegradablepolymer; and a sugar.
 2. The composition of claim 1 in which the sugaris sucrose.
 3. The composition of claim 1 which further comprises asurfactant.
 4. The composition of claim 3 in which the surfactant is apoloxomer.
 5. The composition of claim 1 which further comprises awater-soluble polymer.
 6. The composition of claim 5 in which thewater-soluble polymer is a medium molecular weight polyethylene glycol.7. The composition of claim 1 in which the gas is nitrogen.
 8. Thecomposition of claim 1 in which the biodegradable polymer is selectedfrom the group consisting of polycaprolactone, polylactice,polyglycolide,polylactide-polyglycolide copolymers, copolymers oflactides and lactones, delta-valerolactone, polyalkylcyanoacrylates,polyamides, polyhydroxybutyrates, polydioxanones,poly-beta-aminoketones, polyanhydrides, poly-(ortho)esters, polyglutamicacid, polyaspartic acid and esters of polyglutamic and polyasparticacids.
 9. The composition of claim 1 in which the cross-linkedamphiphilic material is a cross-linked protein.
 10. The composition ofclaim 9 in which the cross-linked protein is cross-linked human serumalbumin.
 11. The composition of claim 1 in which the cross-linkedamphiphilic material is cross-linked with glutaraldehyde.
 12. Acomposition comprising: a plurality of microparticles, a majority ofwhich have diameters in the range of about 1 to 10 micrometers, each ofwhich comprises a shell enclosing a gas-filled hollow core, said shellcomprising an inner layer of polylactide and an outer layer ofglutaraldehyde cross-linked human serum albumin; glycine; andpolyethylene glycol
 3350. 13. The composition of claim 12 which is inthe form of a dry cake and/or powder.
 14. The composition of claim 12which is in the form of an aqueous suspension.